Obesity, diabetes mellitus, and atherosclerotic heart disease are associated with increases in triacylglycerol synthesis. The initial and committed step in this pathway is the acylation of sn-glycerol-3 phosphate to form 1 acyl-sn-glycerol-3-phosphate which is catalyzed by glycerol-3-phosphate acyltransferase GPAT. Since this step is believed to be rate limiting, GPAT is likely to play the pivotal role in regulating triacylglycerol and phospholipid biosynthesis. The specific purpose of this research proposal is to understand the activity, regulation, and requirement for mitochondrial GPAT in the context of its control of glycerolipid biosynthesis. To study GPAT activity, I will use oligonucleotide directed mutagenesis to identify the critical amino acids required for GPAT action. Protease accessibility studies will determine the topography of GPAT in the outer mitochondrial membrane and thereby aid in understanding both activity and regulation. GPAT regulation will be examined directly by determining if it is phosphorylated and inactivated during nutritional states associated with decreased triacylglycerol synthesis. Interactions between GPAT and other enzymes of glycerolipid biosynthesis will be examined through use of the yeast two-hybrid system and cross-linking experiments. Finally, the role of mitochondrial GPAT in directing the fate of fatty acids during different nutritional and hormonal states will be examined in tissue culture systems overexpressing or lacking mitochondrial GPAT.